Selective enrichment of antibodies

ABSTRACT

The invention relates to a process for the selective concentration of immunoglobulins or other proteins that contain an Fc domain (target protein), comprising the following steps:
         a. preparing a solution that contains the target protein;   b. incorporating an Fc-binding protein with precisely two binding sites under conditions that allow binding to occur;   c. separating the precipitate from the liquid phase;   d. undoing the binding of the target protein from the Fc-binding protein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 14, 2020, isnamed 01-2592-US-3-2020-12-16-SequenceListing.txt and is 6,743 bytes insize.

BACKGROUND TO THE INVENTION Technical Field

The present invention relates to purification processes for proteins,particularly immunoglobulins or other proteins that have an Fc domain.

State of the Art

Biomolecules such as proteins, polynucleotides, polysaccharides and thelike are increasingly gaining commercial importance as medicines, asdiagnostic agents, as additives in foods, detergents and the like, asresearch reagents and for many other applications. The need for suchbiomolecules can no longer normally be met—for example in the case ofproteins—by isolating molecules from natural sources, but requires theuse of biotechnological production methods.

The biotechnological preparation of proteins typically begins with thecloning of a DNA fragment into a suitable expression vector. Aftertransfection of the expression vector into suitable prokaryotic oreukaryotic expression cells and subsequent selection of transfectedcells the latter are cultivated in bioreactors and the desired proteinis expressed. Then the cells or the culture supernatant is or areharvested and the protein contained therein is worked up and purified.

In the case of eukaryotic expression systems, i.e. when using mammaliancell cultures such as CHO or NSO cells, for example, there has been asignificant increase in the last 15 years in the concentration of thedesired protein in the cell cultures or cell culture supernatants thatcan be achieved in the expression step. Over the same period, thebinding capacity of chromatographic materials that are used in thesubsequent purification of the proteins increased only slightly, bycomparison. For this reason there is a pressing need for improved,optimised purification methods for biomolecules, particularly proteins,that can be carried out on a large industrial scale.

In the case of biopharmaceuticals, such as for example proteins used asdrugs, e.g. therapeutic antibodies, the separation of impurities is ofmajor importance, in addition to the product yield. A distinction can bedrawn between process- and product-dependent impurities. Theprocess-dependent impurities contain components of the host cells suchas proteins (host cell proteins, HCP) and nucleic acids and originatefrom the cell culture (such as components of the medium) or from theworking up (such as for example salts or dissolved chromatographyligands). Product-dependent impurities are molecular variants of theproduct with different properties. These include shortened forms such asprecursors and hydrolytic breakdown products, but also modified forms,produced for example by deaminations, incorrect glycosylations orwrongly linked disulphide bridges. Also included among theproduct-dependent variants are polymers and aggregates, Other impuritiesare contaminants. This term covers all other materials of a chemical,biochemical or microbiological nature that do not belong directly to theproduction process. Examples of contaminants include viruses that mayoccur in undesirable manner in cell cultures.

In the case of biopharmaceuticals, impurities give rise to safetyconcerns. These concerns are intensified if, as very often happens withbiopharmaceuticals, the therapeutic proteins are administered directlyinto the bloodstream by injection or infusion. Thus, host cellcomponents may lead to allergic reactions or immunopathological effects.In addition, impurities may also lead to an undesirable immunogenicityof the protein administered, i.e. they may trigger an undesirable immunereaction to the therapeutic agent in the patient, possibly leading tolife-threatening anaphylactic shock. Therefore, there is a need forsuitable purification processes by means of which all unwantedsubstances can be reduced to a harmless level.

On the other hand, even in the case of biopharmaceuticals, economicconsiderations cannot be ignored. Thus, the production and purificationmethods used should not compromise the economic viability of thebiopharmaceutical product thus produced.

As working up and processing steps for proteins, (column)chromatographic processes as well as filtration and precipitationprocesses are of crucial importance. Thus, the precise operation ofconcentrating antibodies contains a step of purification by affinitychromatography. Accordingly, numerous methods of column chromatographyand the chromatography materials that can be used in these processes arecurrently known.

Affinity chromatography matrices are used as the stationary phase in theindustrial purification of various substances. Immobilised ligands canbe used for specifically concentrating and purifying substances thathave a certain affinity for the particular ligand used. For theindustrial purification of antibodies (immunoglobulins), particularlythe purification of monoclonal antibodies, immobilised protein A isoften used as the initial purification step. Protein A is a protein withabout 41 kDa of Staphylococcus aureus that binds with high affinity(10⁻⁸ M-10⁻¹² M of human IgG) to the CH₂/CH₃ domain of the Fc region ofimmunoglobulins. In protein A chromatography, immunoglobulins or fusionproteins that have a protein A-binding Fc region from the mobile phasebind specifically to the protein A ligand, which is covalently coupledto a carrier (e.g. Sepharose). Protein A from Staphylococcus aureus(wild-type protein A) and genetically modified recombinant protein A(rec. protein A) interacts through non-covalent interactions with theconstant region (Fc fragment) of the antibodies. This specificinteraction can be used to separate impurities efficiently from theantibody. By altering the pH, the interaction between the antibody andthe protein A ligand can be deliberately stopped and the antibodyreleased or eluted from the stationary phase.

Apart from protein A as affinity ligand, there are many other moleculescurrently known that bind to the Fc fragment. Thus, individual domainsof protein A are used instead of the complete protein (8). Proteinvariants are known, which differ precisely from the B domain of theprotein A, which are suitable for binding Fc-fragment-containingmolecules (16, 17). These different variants differ essentially in themutations that have been inserted in order to increase the stability orbinding affinity. These mutants of the B domain are usually known asZ-domain or protein Z. Besides protein A or protein G, various peptidesare also suitable for selective binding to the Fc fragment (14). Thepresent great interest in affinity ligands for the Fc fragment lead oneto suppose that still more affinity ligands are being found.

Affinity chromatography and particularly the frequently used protein Achromatography are expensive, however, and precisely when there areincreasing product concentrations in the fermenter and large quantitiesof product, there are limits on the chromatographic purificationprocesses that can be carried out. The critical points are: loadingcapacities, number of cycles, process times, pool volumes and quantitiesof buffer. In the future, therefore, alternative purification processeswill be essential. A general overview of conventional purificationstrategies, including affinity chromatography and alternative methods ofaffinity chromatography, can be found in the following articles (7+10).

A more recent method of affinity chromatography uses not a constantlyimmobilised affinity ligand but, to begin with, a solubilised affinityligand that is mixed with the target protein (11). The affinity ligandcarries a fusion tag or a fusion protein which makes it possible tocarry out the immobilisation of the affinity ligand on a solid phasethat takes place in the second step. In the next step, as inconventional affinity chromatography, the target protein is separatedfrom the ligand under suitable conditions and thus eluted from thecolumn.

The invention described here uses affinity precipitation instead ofaffinity chromatography to purify biomolecules. This method appears tohave great potential precisely when used on a larger scale (1, 2).

Affinity precipitation is the most effective method of proteinprecipitation (2). The precipitation of a protein from a solution ingeneral is a well known process that is frequently used. Thus, manyproteins have already been separated from a solution by ammoniumsulphate precipitation from a protein mixture. During thisprecipitation, macromolecules (e.g. proteins) are removed from thesolution and converted into particles. Depending on the difference indensity between the particle and the solution and the particle diameter,this step leads to precipitation. However, this precipitation usuallytakes place non-specifically.

For more selective concentration of a molecule, e.g. a protein,therefore, affinity precipitation was developed. Affinity precipitationmakes use of the selective binding of an affinity molecule to a targetmolecule. Affinity molecules may be for example proteins, peptides,oligonucleotides or small chemical molecules. A distinction is drawn inaffinity precipitation between two principles, first and second orderaffinity precipitation (7). In first order affinity precipitation, boththe affinity molecule and the target molecule have two binding sites, sothat it is possible for a network to form between the two molecules andan affinity complex is formed that sediments at a specific size. Insecond order affinity precipitation, affinity macroligands (AML) areused, for example. In this type of precipitation, the affinity moleculeis bound to a stimulatable substance, usually a polymer.

The stimulatable substance changes its solubility characteristics as aresult of a change in the ambient conditions, such as e.g. a shift inthe pH or temperature, and precipitation occurs. Unlike first orderaffinity precipitation, there is no need for a bifunctional affinitymolecule or for a bifunctional target molecule either.

In the affinity precipitation that is mostly used, the affinity ligandsare at present coupled to polymers or other mediators (15).

The affinity precipitation of molecules containing Fc fragment hasalready been described in a number of studies. The most commonapplication at present is characterised by the use of so-called “smartpolymers”. “Smart polymers” (or stimuli-responsive “intelligent”polymers or affinity macroligands) are polymers that can change theirproperties in response to external influences (physical or chemicalstimuli). These stimuli may be for example changes in pH or temperature(12-13). Usually, smart polymers react to their stimuli withprecipitation in solution. This precipitation can be reversed, after thedesired separation of the supernatant solution, by suitable conditions.Smart polymers can be conjugated with various biomolecules, leading to alarge accumulation of polymer/biomolecule systems that can be used forall kinds of applications. Examples of these biomolecules includeproteins, oligonucleotides and sugars.

Another form of affinity precipitation is the recently described“affinity sinking” method. In this form of affinity precipitation, alinking molecular scaffold is used to bind a number of affinity ligandsto one another. This then makes it possible to form the network requiredfor precipitation. The binding of the affinity ligand to the networkformer may take place both non-covalently and covalently. This methodwas recently described in the patent “Compositions and methods forpurifying and crystallizing molecules of interest” (6). In this, firstof all a solution containing antibodies is mixed with an affinity ligandthat is covalently bound to a crosslinker. No precipitation is observed.Only after the subsequent addition of a coordinating ion or moleculedoes precipitation take place. In a second similar application theaffinity ligand is linked to a biotin-binding protein which forms anetwork with the mediator avidin (9).

U.S. Pat. No. 7,083,943 describes an affinity precipitation in which abinding domain for the target protein is linked to a scaffold domain theamino acid sequence of which is intended to assist the tendency toprecipitation.

Chen and Hoffman (15) describe the affinity precipitation of IgG withpoly(N-isopropylacrylamide)-protein A conjugates. The precipitation ofantibodies with unmodified protein A, however, has not proved effective.

One disadvantage of the methods described in the prior art is thatduring first order affinity precipitation, precipitation is possibleonly via the cross-linking molecule, or in second order affinityprecipitation a stimulatable substance is required. Other disadvantagesof the affinity precipitations that are best known are on the one handa) steric hindrance in which the binding of the target protein islimited by the binding of high-molecular affinity macroligands, b) theresolubilisation of the precipitated polymer complex is slow, c) thenon-specific co-precipitation of impurities, that generally require asecond precipitation step, d) the additional step of binding an affinityprotein to the polymer or to the crosslinker (2-5).

BRIEF SUMMARY OF THE INVENTION

The present invention relates to affinity precipitation using a bindingprotein with two binding sites, which dispenses completely with anadditional fusion protein or a linker molecule. The affinity protein onits own is sufficient for the precipitation, and there is no need for astationary phase. The invention used here also makes it easier torecover the affinity protein.

The invention relates in particular to a process for the selectiveconcentration of immunoglobulins or other proteins that contain an Fcdomain (target protein), comprising the following steps:

-   -   a. preparing a solution that contains the target protein;    -   b. incorporating an Fc-binding protein with precisely two        binding sites under conditions that allow binding to occur;    -   c. separating the precipitate from the liquid phase;    -   d. undoing the binding of the target protein from the Fc-binding        protein.

In another aspect the invention relates to a process in which theFc-binding protein is a dimer of an Fc-binding domain of protein A orprotein G. Preferably, the two monomers of the dimer are linked to oneanother via a disulphide bridge.

In another aspect the invention relates to a process in which the dimeris a homodimer, the monomers of which have the SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO: 3 or a sequence that differs from SEQ ID NO. 1 in 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 aminoacids.

In another aspect the Fc-binding protein is used in a ratio of 0.5-20 inrelation to the target protein. The solution in step a. preferably has apH of 5.5-8. Preferably, the undoing of the binding in step d. takesplace at a pH of 2-4.5.

In another aspect the invention relates to an Fc-binding proteinconsisting of 2 identical sub-units that have the sequence SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, or a sequence that differs from SEQ ID NO. 1in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or20 amino acids, the two sub-units being linked to each other via acovalent bond. Preferably, the covalent bond is a disulphide bond.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: SDS-PAGE of the Z dimer. To demonstrate the dimerisation viacysteines, iodoacetamide or iodoacetamide with dithiothreitol was addedbefore the application to the SDS-PAGE.

Trace Sample 1 recombinant Z-dimer mixed with 0.6 μmol iodoacetamide 2recombinant Z-dimer mixed with 0.3 μmol DTT and iodoacetamide 3 purerecombinant Z-dimer 4 Kombi marker 5 recombinant Z-dimer mixed with 0.2μmol iodacetamide 6 recombinant Z-dimer mixed with 0.1 μmol DTT and 0.2μmol iodoacetamide 7 pure recombinant Z-dimer

FIG. 2: Affinity precipitation using Z-dimer. As the first step, proteinZ is oxidised. In the second step the Z-dimer is added to a solutioncontaining antibody (mAB), which leads to the selective precipitation ofthe antibody.

FIG. 3: UV diagram of ion exchange chromatography under acid conditionsfor separating the Z dimer from the antibody (Experiment 2). The UVchromatogram at 220 nm is shown in red, the UV chromatogram at 280 nm isshown in blue and the increasing content of buffer B (20 mM phosphate, 3M NaCl) is shown in green.

FIG. 4: SDS-PAGE for Experiment 2.

Trace Sample 1 purified antibody 2 protein Z dimer 3 supernatant afterprecipitation 4 supernatant after an optional washing step 5 resuspendedpellet (antibody with Z-dimer) 6 peak 1 of the ion exchangechromatography at an acid pH (antibody) 7 peak 2 of the acid ionexchange chromatography at an acid pH (Z-dimer) 8 Kombi marker

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of depleting impurities,particularly host cell protein (HCP) and DNA, from protein compositionsof the kind obtained from cell cultures in which proteins are expressedrecombinantly or endogenously. In particular, the invention relates tomethods of purifying or concentrating a protein (target protein) bybinding an Fc-binding protein or multimer thereof with at least twobinding sites. In further steps the precipitate is separated off andthen the binding of the Fc-binding protein to the target protein isremoved using suitable conditions.

The present invention relates to a process for the selectiveconcentration of immunoglobulins or other proteins that contain an Fcdomain (target protein), comprising the following steps:

-   -   a. preparing a solution that contains the target protein;    -   b. incorporating an Fc-binding protein with precisely two        binding sites under conditions that allow binding to occur;    -   c. separating the precipitate from the liquid phase;    -   d. undoing the binding of the target protein from the Fc-binding        protein.

The target protein may be in particular an immunoglobulin or a proteinthat contains the Fc domain of an immunoglobulin and can bind to proteinA or fragments of protein A. Immunoglobulins consist of two heavy andtwo light chains. The heavy chains each have one variable and three tofour constant domains depending on the immunoglobulin. These arereferred to analogously as VH and CH1, CH2, CH3. The variable domains ofa light and a heavy chain form the antigen binding site. The domain CH2contains a carbohydrate chain which forms a binding site for thecomplement system. The CH3 domain contains the Fc-receptor binding site.Target proteins to which the process according to the invention can beapplied are all proteins that have an Fc domain. Examples of proteinsthat contain CH2/CH3 regions are antibodies, immunoadhesins and fusionproteins in which the protein of interest is connected to a CH2/CH3region. In one embodiment of the invention, the target protein is forexample an antibody that has a CH2/CH3 region and is thus capable ofbinding to protein A. The term CH2/CH3 region refers to the amino acidsin the Fc region of an antibody that interact with protein A.

The Fc-binding protein comprises according to the invention preciselytwo binding sites for one Fc domain.

In another aspect the invention relates to a process wherein theFc-binding protein is a dimer of an Fc-binding domain of protein A orprotein G. The two monomers of the dimer are preferably linked togetherby a disulphide bridge.

By an Fc-binding protein are meant proteins or peptides which arecapable of binding to the Fc region. Preferably, Fc-binding proteinsbind with a dissociation constant (Ko value) in the range from10⁻²-10⁻¹³M.

In preferred embodiments of the invention the Fc-binding protein is ahomo- or heterodimer of Fc-binding domains which comprise or contain thesequences listed in Table 1:

SEQ ID NO: Sequence Description  1MVDNKFNKEQ QNAFYEILHL PNLNEEQRNA FIQSLKDDPS Z-domain Cys tagQSANLLAEAK KLNDAQAPKS SACRRRRRRR RP  2MVDNKFNKEQ QNAFYEILHL PNLNEEQRNA FIQSLKDDPS Z-domain CysQSANLLAEAK KLNDAQAPKS SAC  3 DNKFNKEQQN AFYEILHLPN LNEEQRNAFI QSLKDDPSQSZ-domain ANLLAEAKKL NDAQAPK  4QQNAFYQvLN MPNLNADQRN GFIQSLKDDP SQSANVLGEA E domain of QKLNDSQAPKprotein A (Swissprot P02967)  5QNNFNKDQQS AFYEILNMPN LNEAQRNGFI QSLKDDPSQS D domain ofTNVLGEAKKL NESQAPK protein A (Swissprot P02967)  6DNNFNKEQQN AFYEILNMPN LNEEQRNGFI QSLKDDPSQS C domain ofANLLSEAKKL NESQAPK protein A (Swissprot P02967)  7DNKFNKEQQN AFYEILHLPN LNEEQRNGFI QSLKDDPSQS B domain ofANLLAEAKKL NDAQAPK protein A (Swissprot P02967)  8DNKFNKEQQN AFYEILHLPN LTEEQRNGFI QSLKDDPSVS A domain ofKEILAEAKKL NDAQAPK protein A (Swissprot P02967)  9TTYKLVINGK TLKGETTTKT VDAETAEKAF KQYANDNGVD protein G Fc-GVWTYDDATK TFTVT binding domain from Streptococcus Sp. (Uniprot Q53337)10 TTYKLVINGK TLKGETTTKA VDAETAEKAF KQYANDNGVD protein G Fc-GVWTYDDATK TFTVT binding domain from Streptococcus dysgalactiae(YP_002997067) 11 TTYRLVIKGV TFSGETATKA VDAATAEQTF RQYANDNGITIgG binding domain GEWAYDTATK TFTVTE from Streptococcus equi(YP_002123072)

By a homodimer is meant, in this context, an Fc-binding protein that ismade up of two sub-units of the same sequence.

By a heterodimer is meant, in this context, an Fc-binding protein thatis made up of two sub-units of different sequences, each of which has abinding site for an Fc domain. Preferably, the sub-units containsequences that are selected from the sequences in Table 1.

In another aspect, the invention relates to a process in which the dimeris a homodimer the monomers of which have SEQ ID NO: 1, SEQ ID NO: 2 orSEQ ID NO: 3 or a sequence that differs from SEQ ID NO. 1 in 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.The monomers that differ from SEQ ID NO. 1 in 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids have theproperty of binding Fc-domains with a K_(D) value in the range from10⁻²-10⁻¹³M.

Binding conditions are conditions in which binding to the target proteinby the Fc-binding proteins takes place, preferably in a pH range of pH5.5-9, preferably 6-8.

The precipitation occurs spontaneously under binding conditions, such asthose found for example in cell-free eukaryotic culture supernatant.There is no need to link the dimers according to the invention topolymers, e.g. polyethyleneglycols, in order to promote precipitationwith polymers.

In another aspect the Fc-binding protein is used in a molar ratio of0.5-20 relative to the target protein.

The separation of the precipitate may be carried out by centrifugationand subsequent removal of the supernatant, but also by filtrationtechniques.

The undoing of the binding to the target protein is carried out underconditions that enable the Fc-binding protein to be separated from thetarget protein. Preferably, this can be done by adjusting the pH to arange between pH 2 and 4.5.

In another aspect the invention relates to an Fc-binding protein thatconsists of 2 identical sub-units that have the sequence SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, or a sequence that differs from SEQ ID NO. 1in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or20 amino acids, wherein the two sub-units are linked together by acovalent bond. Preferably, the covalent bond is a disulphide bond.

Examples

Equipment and Methods:

Preparation of the Dimerised Protein Z

Protein Z was obtained as a recombinant protein from E. coli. Theremoval of impurities was carried out after separation of the celldebris by ion exchange chromatography.

Protein sequence of the Z domain used:

(SEQ ID NO: 1) Met-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Asn-Ala-Phe-Tyr-Glu-Ile-Leu-His-Leu-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Phe-Ile-Gln-Ser-Leu-Lys-Asp-Asp-Pro-Ser-Gln-Ser-Ala-Asn-Leu-Leu-Ala-Glu-Ala-Lys-Lys-Leu-Asn-Asp-Ala-Gln-Ala-Pro-Lys-Ser-Ser-Ala-Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Pro

By inserting a non-native cysteine into the peptide chain of the Zdomain it is possible to deliberately connect two protein Z moleculesvia a disulphide bridge. Directly after the purification of therecombinant protein Z the latter is obtained by oxidation as a dimerisedprotein.

Cell-Free Eukaryotic Culture Supernatant

The cell culture supernatant of CHO cells optimised to secretoryproduction was obtained by filtration or centrifugation after severaldays' culture.

SEC Analysis

The analysis of protein impurities and the antibody content of thesamples from Experiment 1 was carried out by analytical size exclusionchromatography (SEC). A TSK-GEL SW 3000 was used with the precolumnrecommended by the manufacturer (TOSOH Bioscience). Analysis was carriedout on a Dionex Ultimate apparatus with monitoring of the UV signal at280 and 220 nm.

Ion Exchange Chromatography for Separating the Z-Dimer from the Antibody

The ion exchange chromatography was carried out on an AKTA Explorer 10apparatus (GE Healthcare) with observation of the UV absorption at 220and 280 nm. The column material used was SP Sepharose FF (19 mL gel bedvolume). After the application of the pellet resuspended at an acid pH,the column was equilibrated with 20 mM phosphate buffer at pH 3.3 andthen the antibody and the Z-dimer were separated from one another over a25 column volume long gradient. A 20 mM phosphate buffer with 3 M NaClat pH 3.3 was used for the elution.

Protein A HPLC

Protein A HPLC on a Waters or Dionex apparatus (injector pumps andcolumn oven W2790/5, UV detector W2489) was used to determine theantibody content of cell culturefree supernatant and purified antibody.The antibody content of the solutions was determined using the UV signalof the acidically eluting peak.

Concentration

The solutions obtained in the tests were partly concentrated for theanalytical methods (Am icon Ultra, exclusion size 3 kD).

SDS-PAGE:

A 20% homogeneous SDS gel was used to test the individual fractions andsupernatants. The protein bands were detected by silver stainingaccording to Heukeshoven.

Host Cell Protein Analysis by ELISA Assay

In Experiment 3 the host cell protein analysis was carried out using anELISA assay.

DNA Analysis

The DNA analysis was carried out after single strand production by anenzymatically catalysed detecting reaction.

UF/DF

A 50 kDa Centramate Sheet PES (polyethersulphone) made by Messrs Pallwith a surface area of 180 cm² was used for the UF/DF tests inExperiment 3. The UF/DF was carried out so that the antibody wasinitially concentrated six-fold and then diafiltered (exchanged for 6volumes of the buffer) before being circulated for 10 min with theretentate valve fully open. The diafiltration was repeated twice. Thetotal UF/DF was carried out with 50 mM acetate buffer+100 mMarginine+150 mM NaCl pH 3.0.

Hydrophobic Interaction Chromatography (HIC)

The hydrophobic interaction chromatography (Experiment 3) was carriedout with a 27 mL column on an AKTA apparatus. The column material usedwas Toyopearl Phenyl 650 M made by Tosoh. For this purpose, after theUF/DF, 3.5 M ammonium sulphate buffer was added to the retentate until aconductivity of 165 mS/cm was obtained. The column equilibrated withbuffer (50 mM acetate+1.2 M ammonium sulphate pH 4) was charged with theretentate from the UF/DF and then a gradient was run over 40 bed volumesto the buffer 50 mM acetate, pH 4.

Experiments:

Detecting the Dimerisation of Protein Z by Cysteine

Detection of the dimerisation of protein Z by cysteine was carried outby SDS-PAGE analysis (FIG. 1). To ensure that the working up does notinduce any oxidation of the protein Z, first of all the free cysteinegroups were alkylated with iodoacetamide. For detecting the monomer,additionally reduction was carried out with dithiothreitol (DTT). 2.6nmol of Z-dimer were mixed with DTT or a corresponding volume of bufferand incubated for five minutes at 95° C. Then the mixture was cooled toambient temperature and iodoacetamide or a corresponding volume ofbuffer was added and the mixture was incubated for a further 20 min inthe dark. After five minutes' incubation with SDS-PAGE buffer thepreparation was applied to the SDS-PAGE.

The SDS-PAGE analysis shows that the Z monomer is formed by the additionof the reducing agent DTT and the gel band of the Z dimer disappears.

Selective Precipitation Using a Z-Dimer

The tests described below show that the selective precipitation of anantibody can be achieved with a Z-dimer (FIG. 2, Experiment 1). TheZ-dimer is the dimerised B-domain of protein A from Staphylococcusaureus linked via two non-native cysteines, which carries a mutationcompared with the wild-type (cf. Equipment and Methods). Moreover,Experiment 2 demonstrates that the pellet obtained can be put back intosolution by resuspension in the acid pH range and then an antibody canbe removed again from the Z-dimer by ion exchange chromatography at anacid pH.

Experiment 1:

Different ratios of protein Z to antibody that was present in cell-freeeukaryotic culture supernatant were tested. In addition, the volume ofthe individual batches was varied. In test 1, 0.13 μmol of Z-dimer wereincubated, with shaking, with 0.065 μmol of IgG4 contained in cell-freeeukaryotic culture supernatant in a total volume of 5.47 mL for 2 h. Intest 2, 16.25 nmol of Z-dimer were incubated, with shaking, with 16.25nmol IgG1 contained in cell-free eukaryotic culture supernatant in atotal volume of 5.2 mL, again for 2 h. Then the mixture was centrifugedfor 10 min at 4000 rpm and the supernatant was separated from thepellet. The antibody content of the supernatant was determined by meansof the UV signal of analytical SEC and by protein A chromatography(Equipment and Methods). The content of antibody in the pellet wasdetermined by subtracting the antibody content of the supernatant fromthe total content of antibody used. A precipitation of up to 99%antibody was observed (test 1: 99%/test 2: 76%). Moreover, the culturesupernatant was examined for protein impurities before and after theprecipitation by analytical SEC. The chromatogram from the cell-freeeukaryotic culture supernatant was compared directly with thesupernatant after precipitation. Depletions of protein impuritiesranging from 90-99% were observed (test 1 90%/test 2 99%). This meansthat 1% or 10% of the protein impurities had been co-precipitated.Protein impurities here may be both host cell proteins and fragments oraggregates of the target protein.

Experiment 2:

A cation exchanger (SP Sepharose FF) was used to separate the Z-dimerfrom the antibody. 0.2 μmol of purified antibody were incubated with 0.2μmol of Z-dimer in a volume of 10.1 mL for 36 min. After subsequentcentrifugation (4000 rpm, 10 min) the supernatant was removed. Then thepellet was dissolved batchwise in 10 mL phosphate buffer at pH 7.4 andcentrifuged again (4000 rpm, 10 min). The pellet thus obtained wasresuspended in 20 mL phosphate buffer (20 mM phosphate, pH 3.3) andpurified using an ion exchanger. The Z-dimer could be separated from theantibody through a gradient over 25 bed volumes (FIGS. 3 and 4).

Experiment 3:

A multi-step antibody purification process was carried out with affinityprecipitation as the capture step.

500 mg of antibody (3.3 μmol) were used in a ratio of 1:2 to Z-dimer(6.6 μmol) in the precipitation from cell-free culture supernatant.After one hour's incubation with gentle agitation, the entire suspensionwas added to a 0.22 μm filter (Millipore) and in this way thesupernatant of the precipitate was separated by filtration. Theprecipitate was then washed with buffer (231 mL of 50 mM phosphatebuffer, pH 7.4) and in the next step resuspended in the filter withacetate buffer (179 mL 50 mM acetate buffer+100 mM arginine+150 mM NaClpH 3.0). To separate the Z-dimer from the antibody, UF/DF was carriedout with a 50 kDa membrane. The next step was HIC to separate off theZ-dimer and further impurities.

The total yield of the antibody was 98% after the precipitation anddecreased to 87% after UF/DF and to 64% after HIC. In addition to thegood yields obtained with the selective precipitation of the antibody,DNA and host cell protein analysis showed that the DNA could be reducedby 99% by the precipitation step followed by UF/DF and the host cellprotein content could be reduced by 99.9% compared with the initialvalue (cell-free culture supernatant).

LITERATURE

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1-9. (canceled)
 10. Fc-binding protein that consists of 2 identicalsub-units that have the sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, or a sequence that differs from SEQ ID NO. 1 in 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, whereinthe two sub-units are linked together via a covalent bond. 11.Fc-binding protein according to claim 10, wherein the covalent bond is adisulphide bond.
 12. Fc-binding protein according to claim 10, whereinthe Fc-binding protein is a dimer of an Fc-binding domain of protein Aor protein G.
 13. Fc-binding protein according to claim 12, wherein theFc-binding domain comprises or contains one of the sequences SEQ ID NO:1 to SEQ ID NO:
 11. 14. Fc-binding protein according to claim 12,wherein the two monomers of the dimer are linked together by adisulphide bridge.
 15. Fc-binding protein according to claim 10, whereinthe dimer is a homodimer, the monomers of which have the sequence SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a sequence that differs from SEQID NO. 1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 amino acids.